O-fucosylation of an antibody light chain: characterization of a modification occurring on an IgG1 molecule.
We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.
Valliere-Douglass
John F
JF
Department of Analytical and Formulation Sciences, Amgen, 1201 Amgen Court West, Seattle, WA 98119-3105, USA.
Brady
Lowell J
LJ
Farnsworth
Chris
C
Pace
Danielle
D
Balland
Alain
A
Wallace
Alison
A
Wang
Wesley
W
Treuheit
Michael J
MJ
Yan
Boxu
B
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D005643:Fucose